CN113840619A - Cyclodextrin-based injectable co-formulations of SGLT2 inhibitors and incretin peptides - Google Patents

Abstract

Provided herein are co-formulations comprising cyclodextrins that allow for the simultaneous subcutaneous administration of a sodium glucose co-transporter 2 inhibitor (SGLT2i), such as dapagliflozin and an incretin peptide, such as GLP-1/glucagon dual agonist peptide.

Description

Cyclodextrin-based injectable co-formulations of SGLT2 inhibitors and incretin peptides

Reference to electronically submitted sequence Listing

The contents of the electronically-submitted sequence listing of the ASCII text file filed with the present application (title: GLPGG-202-US-PSP _ sl. txt; size: 4,096 kilobytes; and creation date: 2019, 5, 21 days) are incorporated herein by reference in their entirety.

Background

Many complex progressive diseases, including asthma, cancer and diabetes (to name a few), are posing a worldwide disease burden. In order to adequately control the progression of these heterogeneous diseases, combination therapy has proven to be an effective drug treatment strategy. Typically, patients begin to use a single drug to control symptoms and delay disease progression, adding additional drugs as the underlying pathophysiology deteriorates over time and symptoms become more difficult to control.

Type 2 diabetes (T2D) is a metabolic disorder characterized by high blood glucose levels that, if poorly controlled, can lead to life-threatening health complications. If initial intervention of diet and exercise alone is not possible, the antidiabetic drug is started at the time the patient starts the metformin monotherapy treatment. As the disease progresses and blood glucose returns to the diabetic range, additional drugs with different mechanisms of action are added. Finally, T2D patients received a dual or triple therapy containing metformin or insulin as one of the active ingredients of their pharmaceutical "cocktail". This enormous medication burden often results in low compliance.

Non-adherence to diabetes therapy is a recognized challenge and one of the major reasons for patients' failure to control blood glucose. Typically, more than half of patients receiving anti-diabetic therapy have inadequate control, defined as HbAlc levels greater than 7.5%. This is driven by a combination of both potential disease progression and poor compliance. According to clinical data in the uk, less than 15% of patients are able to adhere to their diabetes medication. Adherence rates are associated with the complexity of the regimen and decrease from monotherapy to combination therapy, with the lowest adherence rate associated with combination therapy of oral and injectable drugs.

Two of the latest generation antidiabetic drugs, sodium glucose co-transporter 2 inhibitor (SGLT2i) and incretin agonist, are administered as oral and injectable drugs, respectively. Thus, there is a need for co-formulations that can significantly contribute to increased compliance by providing convenient and simultaneous administration of two drugs that otherwise need to be taken separately (e.g., one orally, the other injected).

Disclosure of Invention

Provided herein are pharmaceutical co-formulations comprising (i) an incretin peptide, including inter alia lipidated incretin peptides, (ii) a sodium glucose co-transporter 2 inhibitor (SGLT2i) and (iii) a cyclodextrin.

In one example, the liquid pharmaceutical composition comprises (i) a lipidated intestinal insulinotropic peptide, (ii) a sodium glucose co-transporter 2 inhibitor (SGLT2i), and (iii) a cyclodextrin.

In one example, the incretin peptide is unilipidated. In one example, the insulinotropic peptide is a GLP-1/glucagon dual agonist peptide. In one example, the incretin peptide is MEDI0382, liraglutide, or somagluteptide.

In one example, SGLT2i is dapagliflozin.

In one example, the cyclodextrin is beta cyclodextrin. In one example, the beta cyclodextrin is hydroxypropyl-beta-cyclodextrin. In one example, the cyclodextrin is sulfobutyl ether cyclodextrin.

In one example, the lipidated incretin peptide is present at a concentration of about 0.5 mg/mL. In one example, SGLT2i is present at a concentration of about 17 mg/ml. In one example, the cyclodextrin is present at a concentration of about 7% w/v.

In one example, SGLT2i and cyclodextrin have a stoichiometry of about 1: 1.

In one example, the pH of the composition is from about 6.5 to about 8. In one example, the pH of the composition is from about 7 to about 8. In one example, the pH of the composition is about 7.

In one example, the composition has a volume of 1mL or less.

In one example, the composition is for parenteral administration. In one example, parenteral administration is subcutaneous administration.

In one example, the composition contains inclusion complexes comprising a lipidated intestinal insulinotropic peptide, SGLT2i, and a cyclodextrin.

In one example, the composition does not comprise fibrils of lipidated intestinal insulinotropic peptides.

In one example, the composition does not reduce the affinity of the reduced insulinotropic peptide for the GLP-1 receptor and/or the glucagon receptor.

In one example, administration of the composition to rats results in a lipidated incretin peptide Cmax of about 390ng/mL, a lipidated incretin peptide Tmax of about 1 hour, a lipidated incretin peptide half-life of about 5 hours, and/or a lipidated incretin peptide AUC of about 3500 and 4000ng.hr/mL_0-inf。

Also provided herein are injection pens comprising any of the compositions provided herein. In one example, the injection pen delivers about 600 μ L of the composition.

Also provided herein is a method of treating type 2 diabetes in a subject in need thereof, the method comprising administering to the subject any of the compositions provided herein. In one example, the subject is overweight or obese.

Also provided herein are methods of treating nonalcoholic steatohepatitis (NASH) or nonalcoholic fatty liver disease (NAFLD) in a subject in need thereof, comprising administering to the subject any of the compositions provided herein. In one example, the subject is overweight or obese.

Also provided herein is a method of reducing liver fat in a subject in need thereof, the method comprising administering to the subject any of the compositions provided herein. In one example, the subject is overweight or obese.

In one example of the method, administration delivers about 10mg of SGLT2i and/or about 300 μ g of lipidated incretin peptide to the patient. In one example, administration is dietary and exercise assistance.

Drawings

FIG. 1 shows the chemical structure of MEDI0382(SEQ ID NO: 4), formula (C)₁₆₇H₂₅₂N₄₂O₅₅) And molecular weight (3728.09).

Fig. 2 provides Dapagliflozin (DPZ): hydroxypropyl- β -cyclodextrin (HP β CD) complex and Engeletin (EPZ): phase solubility profile of HP β CD complex. DPZ and EPZ showed an increase in apparent solubility with increasing HP β CD concentration. (see example 1.)

FIG. 3 shows the results of aggregation kinetics studies of MEDI0382(SEQ ID NO: 4) in various formulations (including in buffer, in 7% HP β CD, and coformulated with DPZ in 7% HP β CD). (A) The figure shows the time course of fibril formation followed by ThT fluorescence intensity measurements. The results show that fibrillation was completely inhibited by the addition of cyclodextrin, both in the presence and absence of DPZ. Data are presented as mean ± SD (n ═ 3). (B) The figure shows secondary structure of MEDI0382 in buffer and 7% HP β CD characterized by far UV circular dichroism before and after incubation at 37 ℃. MEDI0382 in buffer showed a typical α -helical spectrum at T0, with a primary β -sheet structure at Tend (218nm), confirming the presence of fibrils. When formulated in HP β CD, a change in CD spectrum was observed, but remained unchanged throughout the incubation period. (see example 2.)

Figure 4 shows representative TEM images of MEDI0382 before and after incubation at 37 ℃ confirming fibril formation only in the formulation of MEDI0382 in buffer (scale bar 200 nm). (see example 2.)

Figure 5 shows representative TEM images of liraglutide in buffer neutralized HP β CD and related vehicle after Tht assay, confirming fibril formation only in formulations of liraglutide in buffer. (scale bar 200nm) (see example 2.)

Figure 6 shows the liraglutide FTIR spectra after Tht assay. (see example 2.)

FIG. 7 shows the characteristics of MEDI0382-HP β CD interaction. (A) The graph provides a qualitative assessment of cyclodextrin cavity occupancy by ANS fluorescence measurement. Since ANS has the property of fluorescing upon complexation with HP β CD, ANS was used as a probe to assess the extent of free cavity in various formulations (including 7% HP β CD vehicle, MEDI0382 in 7% HP β CD, DPZ in 7% HP β CD, and MEDI0382+ DPZ in 7% HP β CD). Buffer vehicle and MEDI0382 in buffer were used as controls. Lower fluorescence indicates higher occupancy. Data are expressed as mean ± s.d. (n ═ 3). (B) The figure shows the measurement of intrinsic tryptophan (Trp) fluorescence. Trp fluorescence emission spectra provide information on changes in the microenvironment depending on the formulation. MEDI0382+ DPZ formulation was not measured due to interference of DPZ. Data are expressed as mean ± s.d. (n ═ 3). (see examples 3 and 4.)

Figure 8 shows the near UV circular dichroism spectrum of MEDI0382 in buffer and 7% HP β CD. The contribution of aromatic residues is prominent as follows: 285-310nm for tryptophan (Trp), 275-285nm for tyrosine (Tyr), and 255-270nm for phenylalanine (Phe). (see example 4.)

Fig. 9 shows the data corresponding to (a) HP β CD: DPZ and (B) HP β CD: typical ITC isotherm for titration of MEDI 0382. HP beta CD: DPZ is an exotherm, while HP β CD: the titration result of MEDI0382 is an endothermic isotherm. (see example 3.)

FIG. 10 shows the 1H-1H NOESY spectral region of MEDI0382 with 10% HP β CD (NMR water inhibition). (A) The NOESY region focused on the interaction between aromatic residues and HP β CD. (B) Schematic representation of interaction with Trp. (C) The NOESY region focused on the interaction between palm lipid chains. (D) Schematic representation of the interaction. (see examples 4 and 5.)

Fig. 11 shows (a) the start of the docking of HP β CD onto the peptide, MEDI 0382: snapshot after 100ns simulation of HP β CD complex. The lipid chains form inclusion complexes with HP β CD. (B) Quantification of the type of interaction between HP β CD and peptide residues is also shown. Throughout the simulation, specific hydrogen bonds (grey bars) were formed between the HP β CD and the side chain atoms on average, and in many cases water molecules (black bars) were bridging the interaction. (see example 5.)

Figure 12 shows characterization of MEDI0382 at pH 6.5 and pH 8, Trp fluorescence (left) and by far UV circular dichroism in the presence and absence of cyclodextrin. (see example 6.)

Figure 13 shows the aggregation kinetic profile of MEDI0382 followed by Tht fluorescence at pH 6.5 and pH 8 in the presence and absence of cyclodextrin. (see example 6.)

Figure 14 shows AFM and TEM images of MEDI0382 in buffer and in HP β CD at pH 6.5 and pH 8.0 after Tht assay. (see example 6.)

Figure 15 shows the far UV CD spectra of MEDI0382 after Tht assay at pH 6.5 and pH 8 in the presence and absence of cyclodextrin. The composition of the secondary structure is shown in the table (see example 6.)

Figure 16 shows the results of MEDI0382 aggregation kinetics assays at pH 6.5 and pH 8 in the presence and absence of cyclodextrin. (see example 6.)

Figure 17 shows the aggregation kinetic profile of liraglutide followed by Tht fluorescence at pH 6.5 and pH 8 in the presence and absence of cyclodextrin. (see example 6.)

Fig. 18 shows the far UV CD spectra of liraglutide after Tht assay at pH 6.5 and pH 8 in the presence and absence of cyclodextrin. (see example 6.)

Figure 19 shows the in vitro and in vivo performance of the co-formulations. In vitro potency assays for (a) GLP1 receptor (GLP 1R) and (B) glucagon receptor (GluR). Spectra of plasma concentrations of (C) MEDI0382 and (D) dapagliflozin versus time after subcutaneous injection in rats. (see example 7.)

Detailed Description

It should be appreciated that the specific implementations shown and described herein are examples and are not intended to otherwise limit the scope of the present application in any way.

The patents, patent applications, web sites, company names, and scientific literature referred to herein are hereby incorporated by reference in their entirety to the same extent as if each was specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter. Also, any conflict between a definition in the art of a word or phrase and a definition of the word or phrase as specifically taught in this specification shall be resolved in favor of the latter.

I. Definition of

As used in this specification, the singular forms "a", "an" and "the" specifically encompass the plural forms of the terms they refer to, unless the content clearly dictates otherwise. Thus, the terms "a" or "an", "one or more" and "at least one" are used interchangeably herein.

The term "about" is used herein to mean about (approximate), near (in the region of), rough (roughly), or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the upper and lower limits of the numerical values set forth. Generally, the term "about" is used herein to modify a numerical value by a variation of 20% above and below the stated value, unless otherwise stated.

Further, the use of "and/or" herein should be understood as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and/or" as used herein in phrases such as "a and/or B" is intended to include "a and B," "a or B," "a" (alone), and "B" (alone). Also, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following: A. b, and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).

Technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this application relates, unless otherwise defined. Reference is made herein to various methods and materials known to those skilled in the art. Standard references that illustrate the general principles of Peptide Synthesis include w.c. chan and p.d. white, "Fmoc Solid Phase Peptide Synthesis: a Practical Approach [ Fmoc solid phase peptide Synthesis: utility method ] ", Oxford University Press [ Oxford University Press ], Oxford (2004). Furthermore, circumcise Dictionary of Biomedicine and Molecular Biology [ Concise Dictionary of Biomedicine and Molecular Biology ], Juo, Pei-Show, 2 nd edition, 2002, CRC Press; dictionary of Cell and Molecular Biology [ Dictionary of Cell and Molecular Biology ], 3 rd edition, 1999, Academic Press [ Academic Press ]; and Oxford Dictionary Of Biochemistry And Molecular Biology [ Biochemistry And Molecular Biology Dictionary ], revised edition, 2000, Oxford university Press [ Oxford university Press ] provides the skilled artisan with a general Dictionary annotation for many Of the terms used in this disclosure.

Units, prefixes, and symbols are expressed in their accepted form by the Systeme International de units (SI). Numerical ranges include the numbers defining the range. Unless otherwise indicated, amino acid sequences are written from left to right in the amino to carboxyl orientation. The headings provided herein are not limitations of the various aspects of the disclosure which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

The terms "peptide", "polypeptide", "protein" and "protein fragment" may be used interchangeably herein to refer to a polymer of two or more amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers as well as to non-naturally occurring amino acid polymers. The term "peptide" further includes peptides that have undergone post-translational or post-synthetic modifications, such as, for example, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. The "peptide" may be part of a fusion peptide comprising additional components to increase half-life, such as an Fc domain or an albumin domain. Peptides as described herein can also be derivatized in a number of different ways.

The term "amino acid" refers to naturally occurring as well as synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, and those amino acids that are subsequently modified, such as hydroxyproline, γ -carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an alpha carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the chemical structure of a typical amino acid, but that functions similarly to a naturally occurring amino acid. The terms "amino acid" and "amino acid residue" are used interchangeably throughout.

The term "isolated" refers to a peptide or nucleic acid that will generally be in accordance with the state of the present disclosure. Isolated peptides and isolated nucleic acids are free or substantially free of materials with which they are associated in their natural state, e.g., in their natural environment, or other peptides or nucleic acids with which they are present in the environment in which they are prepared (e.g., cell culture) by recombinant DNA techniques practiced in vitro or in vivo. Peptides and nucleic acids may be formulated with diluents or adjuvants and still be isolated for practical purposes-for example if the peptides are to be used to coat microtiter plates for immunoassays, the proteins will typically be mixed with gelatin or other carriers, or pharmaceutically acceptable carriers or diluents when used for diagnosis or therapy.

"recombinant" peptide refers to a peptide produced via recombinant DNA techniques. For the purposes of this disclosure, recombinantly produced peptides expressed in host cells are also considered isolated as are native or recombinant polypeptides that have been isolated, fractionated or partially purified or substantially purified by any suitable technique.

When referring to an insulinotropic peptide, the terms "fragment," "analog," "derivative," or "variant" include any peptide that retains at least some desired activity (e.g., binds to a glucagon and/or GLP-1 receptor). Fragments of the insulinotropic peptides provided herein include proteolytic fragments, deletion fragments that exhibit desirable properties during expression, purification, and/or administration to a subject.

As used herein, the term "variant" refers to a peptide that differs from the recited peptides by amino acid substitutions, deletions, insertions, and/or modifications. Variants can be generated using art-known mutagenesis techniques. Variants may also, or alternatively, contain other modifications-e.g., the peptide may be conjugated or conjugated, e.g., fused to a heterologous amino acid sequence or other moiety, e.g., for increased half-life, solubility, or stability. Examples of moieties conjugated or coupled to the peptides provided herein include, but are not limited to, albumin, immunoglobulin Fc regions, polyethylene glycol (PEG), and the like. The peptide may also be conjugated or coupled to a linker or other sequence (e.g., 6-His) that facilitates synthesis, purification, or identification of the peptide or enhances binding of the polypeptide to a solid support.

The term "pharmaceutical co-formulation" refers to a composition containing an incretin peptide and SGLT2i, and, for example, a pharmaceutically acceptable carrier, excipient, or diluent, for administration to a subject in need of treatment, such as a human subject with type 2 diabetes.

The term "pharmaceutically acceptable" refers to compositions which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity or other complications commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable carrier" refers to one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the incretin peptide and/or SGLT2 i.

An "effective amount" is an amount of the incretin peptide and/or SGLT2i that is administered to a subject in a single dose or as part of a series that is effective for treatment, e.g., for treatment of type 2 diabetes. This amount may be a fixed dose for all subjects being treated, or may vary depending on the weight, health, and physical condition of the subject being treated, the degree of weight loss or weight maintenance desired, and other relevant factors.

The term "subject" means any subject, particularly a mammalian subject, in need of treatment with a pharmaceutical co-formulation provided herein. Mammalian subjects include, but are not limited to, humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, bears, cows, apes, monkeys, orangutans, and chimpanzees, among others. In one example, the subject is a human subject.

As used herein, "subject in need thereof" refers to an individual in whom treatment is desired, e.g., a subject with type 2 diabetes.

Terms such as "treating" or "treatment" refer to therapeutic measures that cure and/or halt the progression of a diagnosed pathological condition or disorder. Terms such as "prevention" refer to prophylactic or preventative measures to prevent and/or slow the development of the targeted pathological condition or disorder. Thus, those in need of treatment include those already suffering from a disease or condition. Those in need of prevention include those susceptible to the disease or disorder as well as those in which the disease or disorder is to be prevented. For example, the phrase "treating a patient with type 2 diabetes" refers to reducing the severity of the disease or disorder to the point where the subject no longer suffers from the discomfort and/or altered function caused thereby. Treatment includes therapeutic measures that slow or alleviate the symptoms of the diagnosed pathological condition or disorder.

As used herein, a "GLP-1/glucagon agonist peptide" is a chimeric peptide that exhibits at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more activity at the glucagon receptor relative to native glucagon, and also exhibits at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more activity at the GLP-1 receptor relative to native GLP-1.

The term "native glucagon" as used herein refers to naturally occurring glucagon, e.g., human glucagon, which comprises the sequence of HSQGTFTSDYSKYLDSRRAQDFVQW LMNT (SEQ ID NO: 1). The term "native GLP-1" refers to naturally occurring GLP-1, e.g., human GLP-1, and is a generic term encompassing, for example, GLP-1(7-36) amide (HAEGTFTSDVSSYLEGQAKEFIAWLVKGR; SEQ ID NO: 2), GLP-1(7-37) acid (HAEGT FTSDVSSYLEGQAAKEFIAWLVKGRG; SEQ ID NO: 3), or a mixture of the two compounds. As used herein, a general reference to "glucagon" or "GLP-1" is intended to mean, without any further designation, native human glucagon or native human GLP-1, respectively. Unless otherwise indicated, "glucagon" refers to human glucagon, and "GLP-1" refers to human GLP-1.

Incretin peptides II

The pharmaceutical co-formulations provided herein comprise an intestinal insulinotropic peptide, including, inter alia, lipidated intestinal insulinotropic peptides. The insulinotropic peptides are agonists of GLP-1, and they include approved GLP-1 single agonists as well as dual or triple agonists, such as MEDI0382, GLP-1/glucagon receptor dual agonists. (see Henderson SJ et al, Diabetes Obes metabes Metab. [ diabetic obesity and metabolism ] 18: 1176-90(2016), incorporated herein by reference in its entirety) lipidation can prolong blood circulation of an incretin peptide. Furthermore, as shown herein, aromatic residues in the lipid chain can interact with cyclodextrins (e.g., HP β CD) in a manner that reduces incretin peptide aggregation.

In one example, the incretin peptide for use in the pharmaceutical co-formulations provided herein is MEDI 0382. MEDI0382 is a toner having HSQGTFTSDX₁₀SEYLDSERARDFVAWLEAGG-a linear peptide of 30 amino acids of the sequence (SEQ ID NO: 4), in which X₁₀Lysine with palmitoyl conjugated to the epsilon nitrogen through a gamma glutamic acid linker (i.e., as described above)K (gE-palmitoyl)). MEDI0382 was palmitoylated to prolong its half-life by binding to serum albumin, thereby reducing its propensity to renal clearance. MEDI0382 has been designed to elicit all positive therapeutic attributes associated with GLP-1 analogues (see Meier jj., Nat Rev Endocrinol. [ natural review of endocrine secretion ]]8: (2012), which is incorporated herein by reference in its entirety), including effective glycemic control, delayed gastric emptying, induction of satiety and weight loss, and the additive effects of glucagon on energy expenditure and metabolic rate. To extend the systemic circulation time of the peptide, a C16 chain was covalently attached to its amino acid sequence, allowing it to reversibly bind to serum albumin. This strategy has previously been successfully applied to liraglutide, an approved GLP-1 peptide single agonist, under the trade name liraglutide

And (4) marketing. In preclinical studies, repeated injections of MEDI0382 resulted in significant weight loss and robust glycemic control in DIO mice and non-human primates. Clinical evaluation is currently underway to treat overweight or obese patients with type 2 diabetes, MEDI0382 has shown efficacy in reducing blood glucose, body weight and liver fat in overweight and obese patients with type 2 diabetes. (see Ambery P, et al, Lancet. [ lancets ]]391: 2607-18(2018), which is incorporated herein by reference in its entirety. )

In one example, the intestinal insulinotropic peptide is MEDI0382, somaglutide, or liraglutide.

Additional incretin peptides can also be used in the pharmaceutical co-formulations provided herein. Exemplary lipidated incretin peptides are provided, for example, in Wang et al, j.control Release [ journal of controlled Release ] 241: 25-33(2016), which is incorporated herein by reference. In certain examples, the lipidated intestinal insulinotropic peptide for use in the pharmaceutical co-formulations provided herein is a mono-lipidated intestinal insulinotropic peptide.

The incretin peptides used in the pharmaceutical co-formulations provided herein may be acylated.

The incretin peptides used in the pharmaceutical co-formulations provided herein can be associated with a heterologous moiety, for example, to prolong half-life. The heterologous moiety is a protein, peptide, protein domain, linker, organic polymer, inorganic polymer, polyethylene glycol (PEG), biotin, albumin, Human Serum Albumin (HSA), HSA FcRn binding moiety, albumin binding domain, enzyme, ligand, receptor, binding peptide, non-FnIII scaffold, epitope tag, recombinant polypeptide polymer, or a combination of two or more of such moieties.

The incretin peptide can be prepared by any suitable method. For example, in certain embodiments, the insulinotropic peptide is chemically synthesized by methods well known to those of ordinary skill in the art, e.g., by solid phase synthesis as described by Merrifield (1963, J.Am.chem.Soc. [ J.Am.Chem.Soc. ] 85: 2149-. Solid phase peptide synthesis can be accomplished, for example, by using an automated synthesizer, using standard reagents, as explained in example 1 of WO 2014/091316.

Alternatively, the incretin peptide can be produced recombinantly using suitable vector/host cell combinations as are well known to those of ordinary skill in the art. A variety of methods are available for recombinantly producing an insulinotropic peptide. Typically, the polynucleotide sequence encoding the insulinotropic peptide is inserted into an appropriate expression vehicle, e.g., a vector containing the necessary elements for transcription and translation of the inserted coding sequence. The nucleic acid encoding the insulinotropic peptide is inserted in the correct reading frame of the vector. The expression vector is then transfected into a suitable host cell expressing the insulinotropic peptide. Suitable host cells include, but are not limited to, bacterial, yeast, or mammalian cells. A wide variety of commercially available host-expression vector systems can be used to express the insulinotropic peptide.

Co-formulation

Provided herein are co-formulations comprising an incretin peptide (as discussed above), a sodium glucose co-transporter 2 inhibitor (SGLT2i), and a cyclodextrin.

SGLT2i is a class of drugs used in conjunction with diet and exercise to lower blood glucose in type 2 diabetic adults. SGLT2i lowers blood glucose by blocking reabsorption of glucose from the kidney. Since this mechanism is independent of insulin and directly related to blood glucose levels, SGLT2i provides a durable method of lowering blood glucose that also minimizes hypoglycemic episodes.

Exemplary SGLT2i include Dacemazin (DPZ), Engagliflozin (EPZ), and canagliflozin. In certain examples, SGLT2i is DPZ or EPZ. In certain examples, SGLT2i is DPZ.

In certain examples, SGLT2i (e.g., DPZ) is present in a pharmaceutical co-formulation provided herein at a concentration of about 17 mg/ml.

Cyclodextrins are cyclic oligosaccharides containing glucopyranose units. Cyclodextrins include alpha, beta, and gamma cyclodextrins, which have varying numbers of glucopyranose units. In certain examples, the cyclodextrin is beta cyclodextrin. An exemplary cyclodextrin is hydroxypropyl- β -cyclodextrin (HP β CD). Another exemplary cyclodextrin is sulfobutyl ether cyclodextrin.

In certain examples, the cyclodextrin (e.g., HP β CD) is present in the pharmaceutical co-formulations provided herein at a concentration of about 7% w/v.

In certain instances of the pharmaceutical co-formulations provided herein, SGLT2i (e.g., DPZ) and cyclodextrin (e.g., HP β CD) have a stoichiometry of about 1: 1.

The pharmaceutical co-formulations provided herein can have a concentration of about 0.5mg/mL of lipidated incretin peptide (e.g., MEDI 0382).

As demonstrated herein, an incretin peptide (e.g., MEDI0382), SGLT2i (e.g., DPZ), and a cyclodextrin (e.g., HP β CD) can be present in an inclusion complex in a pharmaceutical co-formulation provided herein.

The pharmaceutical co-formulations provided herein can have a pH of at least 6.5. The pharmaceutical co-formulations provided herein can have a pH of at least 7.

The pharmaceutical co-formulations provided herein can have a pH of about 6.5 to about 8. The pharmaceutical co-formulations provided herein can have a pH of about 7 to about 8. The pharmaceutical co-formulations provided herein can have a pH of about 7.

The co-formulations may be for parenteral, e.g., subcutaneous, delivery. The co-formulation may be for delivery via a pen device, for example. Accordingly, also provided herein are injection pens comprising the pharmaceutical co-formulations provided herein.

In a co-formulation, SGLT2i and the incretin peptide may share the same injection volume. Pain and tolerance problems can arise in large volume situations. Thus, the co-formulation may have a volume of 1mL or less. Thus, the co-formulation can be designed to be applied in a volume of about 600 μ L. As demonstrated herein, cyclodextrins (e.g., hydroxypropyl- β -cyclodextrin (HP β CD)) can be used as solubility enhancers to accommodate a therapeutically effective dose of SGLT2i in a desired volume.

It is well known that incretin peptides are difficult to formulate due to their inherent self-association and aggregation properties as well as their pH dependent solubility and stability. As demonstrated herein, cyclodextrins (e.g., HP β CD) can be used to prevent aggregation of the intestinal insulinotropic peptide. Thus, the compositions provided herein can lack fibrils of an intestinal insulinotropic peptide (e.g., MEDI 0382). The presence of fibrils can be assessed, for example, using a Transmission Electron Microscope (TEM) or a thioflavin t (tht) assay (e.g., as shown in example 2 herein).

As demonstrated herein, the presence of cyclodextrin and/or SGLT2i with an incretin peptide in a co-formulation does not reduce the efficacy of the incretin peptide (e.g., MEDI 0382). The efficacy of an insulinotropic peptide (e.g., MEDI0382) can be assessed, for example, using in vitro and/or in vivo assays. For example, the activity of an insulinotropic peptide (e.g., MEDI0382) can be assessed based on its activity at the GLP-1 and/or glucagon receptors (e.g., as measured by EC50 in a cAMP accumulation assay, optionally as shown in example 7 herein)

Methods of treatment

The present disclosure provides a method of treating type 2 diabetes comprising administering to a subject in need of treatment a pharmaceutical co-formulation provided herein comprising a lipidated incretin peptide (e.g., MEDI0382) and SGLT2i (e.g., DPZ). In certain examples, administration is dietary and exercise assistance. In certain examples, the subject has 27 to 40kg/m²The BMI of (1). In certain examples, the subject has 30 to 39.9kg/m²The BMI of (1). In some examples, the subject hasThere is a BMI of at least 40. In certain instances, the subject is overweight. In certain examples, the subject is obese.

The present disclosure provides a method of reducing liver fat comprising administering to a subject in need of treatment a pharmaceutical co-formulation provided herein comprising a lipidated incretin peptide (e.g., MEDI0382) and SGLT2i (e.g., DPZ). A reduction in liver fat may lead to increased insulin sensitivity and/or improved liver function. In certain examples, administration decreases hemoglobin A1c (HbA1c) levels. In certain examples, administration is dietary and exercise assistance. In certain examples, the subject has 27 to 40kg/m²The BMI of (1). In certain examples, the subject has 30 to 39.9kg/m²The BMI of (1). In certain examples, the subject has a BMI of at least 40. In certain instances, the subject is overweight. In certain examples, the subject is obese. In certain examples, the subject has type 2 diabetes.

The present disclosure provides a method of treating nonalcoholic steatohepatitis (NASH) comprising administering to a subject in need of treatment a pharmaceutical co-formulation provided herein comprising a lipidated intestinal insulinotropic peptide (e.g., MEDI0382) and SGLT2i (e.g., DPZ). In certain examples, administration is dietary and exercise assistance. In certain examples, the subject has 27 to 40kg/m²The BMI of (1). In certain examples, the subject has 30 to 39.9kg/m²The BMI of (1). In certain examples, the subject has a BMI of at least 40. In certain instances, the subject is overweight. In certain examples, the subject is obese. In certain examples, the subject has type 2 diabetes.

The present disclosure provides a method of treating non-alcoholic fatty liver disease (NAFLD) comprising administering to a subject in need of treatment a pharmaceutical co-formulation provided herein comprising a lipidated intestinal insulinotropic peptide (e.g., MEDI0382) and SGLT2i (e.g., DPZ). In certain examples, administration is dietary and exercise assistance. In certain examples, the subject has 27 to 40kg/m²The BMI of (1). In certain examples, the subject has 30 to 39.9kg/m²The BMI of (1). In certain examples, the subject has a syndrome ofA BMI of 40 less. In certain instances, the subject is overweight. In certain examples, the subject is obese. In certain examples, the subject has type 2 diabetes.

The present disclosure provides a method of treating obesity or an obesity-related disease or disorder, reducing body weight, reducing body fat, preventing weight gain, preventing fat gain, promoting weight loss, promoting fat loss, treating a disease or condition caused by or characterized by excess body weight or excess body fat, managing body weight, improving glycemic control, or achieving glycemic control, wherein the method comprises administering to a subject in need of treatment a pharmaceutical co-formulation provided herein comprising a lipidated incretin peptide (e.g., MEDI0382) and SGLT2i (e.g., DPZ). In certain examples, administration is dietary and exercise assistance. In certain examples, the subject has 27 to 40kg/m²The BMI of (1). In certain examples, the subject has 30 to 39.9kg/m²The BMI of (1). In certain examples, the subject has a BMI of at least 40. In certain instances, the subject is overweight. In certain examples, the subject is obese. In certain examples, the subject has type 2 diabetes.

Examples of other obesity-related (overweight-related) diseases include, but are not limited to: insulin resistance, glucose intolerance, pre-diabetes, elevated fasting glucose, type 2 diabetes, hypertension, dyslipidemia (or a combination of these metabolic risk factors), glucagonomas, cardiovascular disease such as congestive heart failure, arteriosclerosis, atherosclerosis, coronary heart disease, or peripheral arterial disease; stroke, respiratory dysfunction, or kidney disease.

In certain examples, the route of administration of the pharmaceutical co-formulations provided herein comprising lipidated intestinal insulinotropic peptides (e.g., MEDI0382) and SGLT2i (e.g., DPZ) is parenteral. In certain examples, the route of administration of the pharmaceutical co-formulations comprising lipidated intestinal insulinotropic peptides (e.g., MEDI0382) and SGLT2i (e.g., DPZ) provided herein is subcutaneous. In certain examples, the pharmaceutical co-formulations provided herein comprising a lipidated incretin peptide (e.g., MEDI0382) and SGLT2i (e.g., DPZ) are administered by injection, such as from a pen. In certain examples, the pharmaceutical co-formulations provided herein comprising a lipidated intestinal insulinotropic peptide (e.g., MEDI0382) and SGLT2i (e.g., DPZ) are administered by subcutaneous injection.

In certain examples, the pharmaceutical co-formulations provided herein comprising a lipidated intestinal insulinotropic peptide (e.g., MEDI0382) and SGLT2i (e.g., DPZ) can be administered once daily. In certain examples, a pharmaceutical co-formulation provided herein comprising a lipidated intestinal insulinotropic peptide (e.g., MEDI0382) and SGLT2i (e.g., DPZ) can be administered by injection (e.g., subcutaneously) once per day. In certain examples, a pharmaceutical co-formulation provided herein comprising a lipidated incretin peptide (e.g., MEDI0382) and SGLT2i (e.g., DPZ) can be administered by injection (e.g., subcutaneously) once daily for a period of at least one week, for a period of at least two weeks, for a period of at least three weeks, or for a period of at least four weeks.

Examples of the invention

Material

HPLC water and acetonitrile were purchased from VWR (VWR corporation, radner (Radnor), pa). Dapagliflozin is supplied by astrikang (AstraZeneca).

HPB (2-hydroxypropyl-. beta. -cyclodextrin) is supplied by Roche (Roquette Fresh, Lestren, France).

(sulfobutylether-beta-cyclodextrin) was supplied by the company Rieger blue Pharmaceuticals (Ligand Pharmaceuticals, san Diego, Calif., USA). 8-anilino-1-naphthalenesulfonic Acid (ANS) and Thioflavin t (ThT) were purchased from Sigma Aldrich (Sigma-Aldrich), St.Louis, Missouri, USA). Disodium hydrogen phosphate heptahydrate and sodium dihydrogen phosphate monohydrate were manufactured by j.t. becco (j.t. baker chemical co), philippisburg (phillips burg), njWest, usa).

Solubility screening

Dapagliflozin (DPZ) was weighed into a glass vial. The appropriate aqueous vehicle was added to the powder to reach a final concentration of 17mg/mL, vortexed and sonicated. The pass/fail criteria for the formulation were determined by visual observation.

Solubility of DPZ phase in HP β CD (Kleptose HPB)

Solutions of various HP β CDs (Kleptose HPB, roche) were prepared in water at increasing concentrations of 5% to 20% (w/v). Briefly, HP β CD was weighed into a volumetric flask and purified water was added until 80% (v/v) of the final volume. The flask was mixed until completely dissolved and made up to final volume with purified water. About 30mg of DPZ was weighed into each HPLC glass vial, and 500uL of the appropriate HP β CD solution and magnetic flash (magnetic flash) were added thereto. Each concentration was performed in duplicate. The formulation was left for 21 hours and 40 minutes under magnetic stirring. Each sample was then transferred to a 1.5mL microcentrifuge tube and centrifuged at 13,000rpm for 10 minutes. Then 200uL of the supernatant was taken and centrifuged again at 13,000rpm for 30 minutes in a 1.5mL microcentrifuge tube. Finally the samples were diluted in buffer a (95% HPLC water/5% ACN + 0.03% TFA) and the concentration was measured by UPLC according to a calibration curve validated by quality control.

Isothermal titration calorimetry

Isothermal Titration Calorimetry (ITC) measurements were performed at 25 ℃ by titrating cyclodextrins into the peptide or DPZ solution using Microcal Auto ITC 200 (Malvern). MEDI0382 and DPZ solutions were prepared at 0.13mM and 0.12mM, respectively, and cyclodextrin (HP β CD) was prepared at 3mM in match buffer. The experiment was performed in triplicate and each run included 20 injections of 2uL (only 0.4uL for the first injection) with the agitation speed set at 750 rpm. The isotherms were fitted by Malvern Origin software using a set of binding site models.

Fluorescence of tryptophan

Fluorescence measurements were performed on an F-7000FL spectrophotometer at room temperature. 100 μ L of peptide formulation was added in triplicate to 96-well plates (half area). The excitation wavelength was set at 277nm to selectively excite tryptophan fluorescence. Fluorescence emission spectra were scanned between 285nm and 385 nm. Both excitation and emission slits were set at 2.5 nm. Each spectrum is the average of three scans.

Circular Dichroism (CD)

Circular dichroism spectra of freshly prepared peptide solutions at 0.5mg/mL in 20mM sodium phosphate (NaP) buffer (pH 7.0) or in 7% HP. beta. CD/20mM NaP buffer (pH 7.0) were obtained at room temperature using a Jasco J-815 spectrophotometer. Far UV CD data was collected from 180nm-260nm using a 0.1mm path length cuvette and the spectra were deconvoluted using the CONTINLL, SELCON3 and CDSSTR algorithms using CDPro software. Near UV CD data was collected from 250nm to 350nm using a cuvette with a 1cm path length.

Kinetics of aggregation

For aggregation kinetics experiments, MEDI0382 was monitored throughout the thioflavin t (tht) binding assay and compared in the presence and absence of cyclodextrin. Fluorescence measurements were carried out on a Fluostar Optima microplate reader (BMG Labtech, Ofenburg, Offenburg, Germany) thermostatted at 37 ℃. Binding of ThT to fibrils was monitored by using an excitation filter at 440nm and recording the emitted fluorescence at 480 nm. The formulations tested were 20mM NaP buffer pH 7.0 (with and without 7% w/v cyclodextrin). MEDI0382 was formulated to 0.5mg/mL and DPZ was formulated to 17 mg/mL. Mu.l of the formulation was pipetted into the well (to which 10uL of 0.5mM aqueous ThT solution was added) of a 96-well half-area plate (Corning 3881, USA) made of black polystyrene. Each sample was prepared in triplicate. Sealing tape and sealing foil (Costar thermometer protective tube) were used to prevent evaporation. Plate bottom reads were performed every 30 minutes with 5 minutes shaking before each measurement. Each cycle was performed with an orbital shaker at 350rpm, 5 flashes/hole.

NMR

2D NOESY NMR spectra were obtained under water inhibition from 4.3mg/ml MEDI0382 solution with or without 10% HPpCD in NaP buffer (pH 7.6). All NMR experiments were run on a 600MHz Bruker Avance-III HD NMR spectrometer (Bruker-Biospin) equipped with a 5mm TCI cryoprobe at a temperature of 300K using a standard pulse sequence from the Bruker library (TopSpin 3.5). Phase-sensitive NOESY experiments (pulse program "noesyesgpph") were obtained using a stimulated sculpture method for solvent suppression (Hwang T.L SAJ. journal of Magnetic Resonance, Series A. [ Series A ]112 (2): 275-9 (1995)). Spectra were collected under the following conditions: relaxation delay of 1.5s, using 4K 512 data points, in States-TPPI mode (Dominique Mark Mk, et al, Journal of Magnetic Resonance [ J.M2 ]: 393-9(1989)) at a spectral width of 10ppm, acquisition times of 0.341s and 0.043s in F2 and F1, respectively (zero-filling to 1K in F1). 128 scans and 16 false scans were collected per F1 increment, with a mixing time of 0.15 s. Data were processed using Topspin 3.5 software (brueckbaiberg guest) and a sine-bell squared window function (sine-bell squared window function) was applied prior to fourier transformation in the F1 and F2 dimensions.

Transmission Electron Microscope (TEM)

MEDI0382 formulations before and after incubation at 37 ℃ were adsorbed onto 400 mesh copper/carbon film grids (EM resolution), washed twice with deionized water, and then negative-stained with 1.5% uranyl acetate in deionized water. The samples were observed in a FEI Tecnai G2 electron microscope (Thermo Fisher Scientific) operating at 120keV using a 20 μm objective aperture to improve contrast. The images were taken using an AMT camera.

Molecular modeling

Molecular Dynamics (MD) simulations were performed using Desmond software (ACM/IEEE Supercomputing Conference discourse set (SC06) (Proceedings of the ACM/IEEE Conference on Supercomputing (SC06)), Tampa (Tampa), Florida, 2006, 11/11-17 days. The initial geometry is generated for each case as described below. Construction of MEDI0382 peptide (Sturm NS, et al, J Med Chem. [ journal of medicinal chemistry ] using the x-ray structure of glucagon analog (PDB code 1BH0)]41(15): 2693-700(1998)). Manual mutation of amino acids using peptide editing tools in Maestro (Schachleria Schachalalia publication 2018-1: Jaguar: (A))

Release 2018-1: jaguar), Schachoa Corp: (Jaguar)

LLC), new york state, 2018). Deletion of amino acids towards the C-terminus was constructed without template. The side chain of Tyr10 was removed and artificially replaced with the K (. gamma.E-palmitoyl) C-16 fatty acid model. All carboxylic acids, except the protonated C-terminus, remain charged. The final 3D model of MEDI0382 was allowed to relax in NPT Molecular Dynamics (MD) simulations for 10 ns. The equilibrium model was used as a starting point for all further simulations. The 3D model of HP β CD was established based on the x-ray structure of β -cyclodextrin (β -CD) extracted from the CSD database (BCDEXD10) (Klaus Lindner WS, Carbohydrate Research]99(2): 103-15(1982). Four sets of 2-hydroxypropyl groups were manually added to the original β -CD structure. This geometry is relaxed to the nearest energy minimum. A 3D model of the relaxation of HP β CD was used as the starting geometry for all further studies.

The peptide homology model was inserted into a system containing 50000 TIP3 moisture models with

And (6) simulating a box. By adding Na⁺The ions neutralize the system. The peptide concentration was set to 0.55mM and the sodium concentration was set to 2.76 mM. All simulations used OPLS3 force fields against peptides, cyclodextrins and Na ions (OPLS3e, Schachyman Corp.) (

Inc.), new york, state 2013 (Shivakumar D, et al, J Chem Theory company [ journal of chemical Theory and calculation ]]8(8): 2553-8 (2012)). The initial system is allowed to go through a relaxation sequence: a)100ps brownian dynamics NVT ═ 10K; b) limiting the solute heavy atom with small time step length by 12ps NVT MD T (machine direction test) which is 10K; c) for solute heavy atoms, 12ps NPT MD T ═ 10K limits; d) for solute heavy atoms, 12ps NPT MD T-300K limit; and e) NPT MD T ═300K is not limited. After the relaxation protocol, the production simulation was started using the NPT set, the temperature was kept at 300K with the call of a Nose-Hoover chi thermostat (relaxation time of 1ps), and the pressure was kept at 1 atm with the call of a Martyna-Tobias-Klein barometer (relaxation time of 2 ps). The RESPA algorithm is used to integrate the equation of motion at time steps of 2 fs.

In vitro potency assay

CHO-K1 cell lines stably expressing GLP-1 or GCG receptors were stably transduced with cAMP responsive elements linked to luciferase reporter genes to determine in vitro agonist potency of MEDI0382 in buffer, in cyclodextrin and co-formulated with DPZ. Briefly, cells were plated in 96-well white microtiter plates (corning, usa) at 20,000 cells per well and incubated with serially diluted peptide samples for 4 hours before lysis and measurement of cAMP-dependent luciferase activity using Steady-Glo luciferase substrate (Promega, usa). Plates were read on a SpectraMax Paradigm plate reader (Molecular Devices, usa) and 10-point concentration-response curves were generated in triplicate. After performing the parallelism test using SoftMax Pro software (molecular instruments, usa), the results were expressed as the relative potency of the test sample compared to the reference ligand by calculating the ratio of the reference and sample EC50 values from the 4-PL fit, and the reported data are the average of two independent determinations. The fitted curves were analyzed using nonlinear regression in GraphPad Prism software 6.03(GraphPad, usa).

MEDI0382 and DPZ formulations for PK studies

For PK studies, MEDI0382 alone in buffer was prepared at 0.5mg/mL in 50mM sodium phosphate buffer (pH 7.8) + 1.85% Propylene Glycol (PG) (j.t. becker). This buffer allows PK profiles to be compared to historical data.

The cyclodextrin vehicle used for the PK study was 7% w/v HP β CD in 50mM sodium phosphate (pH 7.8) + 0.5% v PG. PG was added to adjust the osmolality of the formulation to 260 mOsm. Briefly, DPZ was dissolved at a concentration of 5mg/mL in (50mM NaP buffer (pH 7.8) + 7% w/v HP. beta. CD in 0.5% v/v PG) vehicle, followed by the addition of MEDI0382 to achieve a concentration of 0.5 mg/mL. In parallel, MEDI0382 alone in buffer was prepared at 0.5mg/mL in 50mM NaP buffer (pH 7.8) + 1.85% v/v PG. The formulations were then diluted to 1/10 with their respective vehicle.

The dose for the PK study was set at 0.5mg/kg and 0.05mg/kg for DPZ and MEDI0382, with a dose volume of 1 mL/kg.

Each group included three animals and serial blood sampling was performed at 0.5, 1, 2, 4, 7, and 24 hours post-dose for PK assessment. Both MEDI0382 and DPZ were analyzed from plasma samples using a validated method (consisting of plasma protein rapid sample preparation followed by LC-MS/MS).

Example 1: cyclodextrin increases solubility of SGLT2i

DPZ recommended dose is 10mg tablets, once daily, for monotherapy and in combination therapy with other hypoglycemic drugs (recommended by the European drug administration (EMA)). Considering that DPZ was well absorbed (reaching 78% absolute bioavailability) after oral administration and that similar exposure was expected for subcutaneous injections, the DPZ dose of the co-formulation was fixed at 10 mg. Thus, the screening assay was designed to target a concentration of 17mg/mL, corresponding to a 10mg dose in a 600. mu.L dose volume. Excipients are selected based on several criteria (e.g., approval status for subcutaneous administration, compatibility with the peptide, and/or priority for increasing solubility of DPZ). Excipients screened included PEG400, PG, DSPE-PEG 2000, glycerol, Kolliphor 188, HP β CD, and BSA. Most excipients do not achieve the desired concentration or maintain DPZ in solution. Only formulations containing cyclodextrins were successful and were therefore further evaluated as potential co-formulation vehicles.

To better understand the enhancing ability of cyclodextrins, we performed a phase solubility study of DPZ in HP β CD (fig. 2). Experimentally the water solubility of DPZ was measured to be 1.6 mg/mL. Upon addition of HP β CD, the solubility of DPZ increased linearly with increasing HP β CD concentration, indicating the formation of a 1: 1 stoichiometric inclusion complex. Binding constant determined by linear regression was 4.7x10³M^-1. According to the phase solubility diagram, required to increase the solubility to 17mg/mLThe amount of HP β CD was 5.5% (w/v). However, for solution formulations it is recommended not to exceed 80% of the saturation solubility to ensure stability, which is set at a level of 7% w/v. To confirm that HP β CD is applicable to other SGLT2i formulations, a phase solubility profile was performed with Engeletin (EPZ), which also shows a linear increase in solubility in the presence of HP β CD (fig. 2).

Example 2: HP beta CD inhibits MEDI0382 aggregation and induces conformational changes

The tendency of peptides such as MEDI0382 to aggregate is one of the major problems in the development of peptide formulations. To assess the physical stability of MEDI0382 in co-formulations, aggregation kinetics studies were performed using the thioflavin t (ThT) assay, which relies on the property of ThT dyes to emit highly enhanced fluorescence upon binding to fibrils (Biancalana M et al, Biochim biophysis Acta [ biochem biophysics ]1804 (7): 1405-12 (2010)). The ThT assay was used to compare aggregation of MEDI0382 at 37 ℃ under various formulation conditions, including cyclodextrin and no cyclodextrin and in the absence or presence of DPZ (fig. 3A). The concentration of MEDI0382 was set at 0.5mg/mL, which corresponds to a clinical dose of 300 μ g.

Prior to the aggregation test, the secondary structure of the freshly prepared formulation was analyzed by far UV CD (fig. 3B) and TEM photographs were taken (fig. 4). MEDI0382 solution in buffer showed CD spectral features of alpha-helical structure with one positive band at 192nm and 2 negative bands at 207nm and 222nm (fig. 3B). Deconvolution of the spectra with CDpro confirmed the presence of a large fraction of the α -helical conformation (51%) and a small fraction of the β -sheet structure (11%). (see table 1 below.) when formulated into HP β CD, significant structural modification was observed with a positive band at 190nm with lower intensity than in the buffer, a negative band at 203nm, and a low intensity band at 224 nm. Determination of secondary structure using CDPro showed that helicity loss was reduced to 18%, which could be compensated by an increase in β -sheets and random coil. (please see table 1 below.)

Table 1: MEDI0382 CDPro

Due to DPZ with a chiral center, the CD spectrum of the co-formulation could not be obtained. In all three MEDI0382 formulations, the absence of fibrils was confirmed by TEM photographs (fig. 4).

Aggregation kinetics of MEDI0382 in buffer, in cyclodextrin, and in co-formulation with DPZ in HP β CD were then monitored by ThT fluorescence measurements (fig. 3A). The ThT spectrum of MEDI0382 in buffer is a sigmoidal curve, indicating that fibrillation has an initial lag phase of 50 hours followed by an elongation phase that appears to plateau towards the end of the assay. TEM pictures taken at the last time point confirmed the presence of fibrils (fig. 4). The far UV CD spectrum after ThT measurement shows that the β -sheet content of the fibrils increases as expected, but it also shows a reasonably high percentage of helicity indicating the structure of the fibrils.

Interestingly, complete inhibition of fibrillation was observed during the assay after addition of cyclodextrin. The absence of fibrils on the TEM images (fig. 4) and the unchanged CD spectrum after ThT assay (fig. 3A) rule out the possibility of false negatives due to the presence of cyclodextrins, and confirm the inhibitory effect of the macrocyclic molecules. Interestingly, the ability to limit fibril growth was also observed for another lipidated GLP1 analogue, liraglutide (fig. 5 and 6).

Finally, co-formulations containing MEDI0382 and DPZ in HP β CD vehicle at pH 7 were also subjected to ThT assays to assess the effect of the presence of DPZ on the physical stability of the peptides. Interestingly, DPZ did not interfere with the inhibitory effect of cyclodextrin, as confirmed by TEM photographs, and fibrillation did not occur (fig. 4).

To determine the mechanism behind the aggregation inhibition of HP β CD, thorough characterization was performed to assess the interaction between the macrocycle and the active molecule.

Example 3: cyclodextrins form inclusion complexes with MEDI0382 and DPZ

ANS is an amphiphilic dye that preferentially binds to hydrophobic cavities and whose fluorescence depends on its environment. In a polar environment, the fluorescence yield remains low, but increases upon interaction with hydrophobic surfaces. ANS can form inclusion complexes with cyclodextrins (Nishijo J, et al, J Pharm Sci. [ J. Pharmatology ]80 (1): 58-62(1991)), and thus it was used to qualitatively compare hydrophobic cores available in various formulations (FIG. 7A). When added to the cyclodextrin vehicle, ANS fluorescence is greatly enhanced compared to the buffer due to interaction with the cyclodextrin cavity. Interestingly, when DPZ or MEDI0382 were formulated with cyclodextrins, the fluorescence intensity decreased, indicating that both drug molecules formed inclusion complexes with cyclodextrins, resulting in a decrease in the available hydrophobic surface for ANS probes. In the cyclodextrin formulation, the lowest intensity was observed for the co-formulation of DPZ and the peptide, indicating that the two molecules are able to interact with the cyclodextrin despite the presence of the other (fig. 7A). ANS was also incubated with MEDI0382 in buffer, but the signal remained low because the peptide comprised mainly alpha helical structures and therefore had a low hydrophobic surface (fig. 7A).

The complexation with HP β CD was further characterized by isothermal titration microcalorimetry experiments (ITC) (fig. 9). ITC is a label-free technique that can determine thermodynamic parameters of biomolecular interactions by measuring the amount of heat released or absorbed during binding. (see Claveria-Gimeno R, et al, Expert Opin Drug discovery]12: 363-77(2017) and Klebe g., Nat Rev Drug Discov [ natural review Drug discovery]14: 95-110(2015)). ITC has become an increasingly preferred technique for characterizing cyclodextrin-guest interactions due to its high sensitivity over the last decade. This high sensitivity enables the measurement of dissociation constants in the millimolar to nanomolar range (Bouchemal K, et al, Drug Discov Today's Drug discovery)]17(11-12): 623-9(2012)). Thus, this method was used to study the interaction between MEDI0382 and HP β CD and compared to DPZ with HP β CD. Two opposite thermodynamic spectra were obtained from titration of HP β CD into DPZ or the peptide (fig. 9). HP beta CD: DPZ exhibit an exothermic spectrum in which the enthalpy and entropy contributions are equal, indicating favorable hydrogen bonding and hydrophobic interactions. By ITC (6.6x 10)³M^-1) Measurement ofDPZ of (c): affinity constant vs. phase solubility plot for HP β CD complex (4.7x 10)³M^-1) The calculated values are very consistent. The 1: 1 stoichiometry of HP β CD: DPZ, previously determined by phase solubility plots, was confirmed by ITC, as shown in Table 2 below.

In contrast to DPZ, HP β CD: the interaction of MEDI0382 appears to be endothermic, characterized by entropy-driven interactions dominated by hydrophobic interactions. Curve fitting of the ITC measurements indicated a stoichiometry of 3: 1. In contrast, titration was also performed with glucagon as well as the non-lipidated analog of MEDI 0382. In both cases, no thermodynamic signal was observed, suggesting that the lipid chain is a key driver of the interaction between cyclodextrin and MEDI 0382.

Table 2: HP beta CD: MEDI0382 and HP β CD: DPZ thermodynamic parameters of ITC interaction

Example 4: HP β CD forms complexes with MEDI0382 through interactions with aromatic residues and lipid chains

To gain insight into the interaction between HP β CD and MEDI0382, near UV CD analysis was performed on the formulations. Since near UV CD is driven primarily by the aromatic chromophores tyrosine (Tyr), phenylalanine (Phe) and tryptophan (Trp), signal changes can provide information about its microenvironment. The spectra of MEDI0382 in buffer and MEDI0382 in cyclodextrin showed a different absorption pattern for each aromatic amino acid region (fig. 8), with Trp being the most affected. This suggests that the local environment of all three chromophores has changed due to secondary structural changes and/or direct interactions with HP β CD.

To further assess the interaction with Trp, intrinsic Trp fluorescence was monitored to provide information about post-formulation changes in cyclodextrins (fig. 7B). Measurements were made with 0 and 7% cyclodextrin. The measured solid tryptophan fluorescence maximum at 342nm in buffer indicates a solvent-exposed Trp residue as previously reported for denatured proteins such as glucagon (352nm) and melittin (346nm) (ghisaidobe AB, et al, Int J Mol Sci. [ international edition of molecular science ] 15: 22518-38 (2014)). Interestingly, an approximately 2-fold increase in Trp fluorescence intensity was observed when formulated in cyclodextrins. The lower fluorescence intensity in the buffer may be due to a quenching effect resulting from exposure of Trp to water (Muino PL, et al, J Phys Chem B. [ physicochemical reports ] 113: 2572-7(2009)), whereas the increase in fluorescence may be related to a shift to a less polar environment (such as the cavity of HPB). Similar enhanced fluorescence emission was observed for free Trp molecules in the presence of cyclodextrins. This observation indicates that there is an interaction between HP β CD and the Trp residue of MEDI 0382.

To confirm the interaction sites between cyclodextrin and peptide, 2D NOESY NMR analysis was performed on MEDI0382 cyclodextrin formulations compared to MEDI0382 in buffer. 2D NOESY NMR spectra were obtained from 4.3mg/ml MEDI0382 solution with or without 10% HP β CD in buffer under water inhibition. As compared to the formulation, MEDI 0382: the proportion of cyclodextrin must be reduced to avoid dynamic range problems in the NOESY NMR spectrum. Due to the insensitivity of NMR techniques, the concentration of MEDI0382 was greatly increased (about 10 times higher than the formulation), while the amount of cyclodextrin was only slightly increased to avoid dynamic range problems and mask the peptide NMR signal. CD analysis confirmed that secondary structure is also affected by the conversion from alpha helix to beta sheet despite the altered ratio. The NMR spectrum of the peptide was previously verified. However, in the current solutions, all amino acids have not been fully assigned. Cyclodextrin resonance assignment is based on literature values (Schneider et al Chemical Reviews [ review of chemistry ], 1998, Vol.98, No. 5). The spectrum of MEDI0382 in the presence of HP β CD showed strong interactions between the H-5 and H-6 protons of HP β CD (FIG. 10B) and several protons from the peptide. The cross-over peaks at about 7.55ppm, 7.40ppm, 7.1ppm, 7.05ppm in F2 and 3.80ppm in F1 (FIG. 10A) indicate NOEs between H5/H-6 of HP β CD and the aromatic protons 43, 40, 41, 42 of Trp (FIG. 10B). The cross peaks at about 7.25ppm in F2 and 3.80ppm in F1 (FIG. 10A) indicate NOEs between H5/H-6 of HP β CD and the aromatic proton 36 of Trp and the aromatic proton of Phe. Another NOE was observed at 3.75ppm in F2 and 1.20ppm in F1 (fig. 10C), indicating a NOE between the cyclodextrin and the lipid chain (fig. 10D).

Example 5: the fibrillation inhibition mechanism is driven by steric hindrance preventing accumulation of II-II and preventing electrostatic attraction and lipid interaction

The stabilizing effect of cyclodextrins on peptides has previously been reported in the literature for insulin, Amyloid-beta and glucagon (see Kitagawa K, et al, Amyloid. [ powdered protein ] 22: 181-6 (2015); Matilainen L, et al, J Pharm Sci. [ J. Med. Sci ] 97: 2720-9 (2008); and Ren B, et al, Phys Chem Phys. [ J. Phys. Physics and Physics ] 18: 20476-85 (2016)). However, the effect can only be demonstrated by delaying the lag time by a few hours or reducing the amount of fibrils; although similar proportions of peptides were used in the case of insulin and glucagon: cyclodextrins, but complete inhibition was not achieved. This difference may be due to the fibrillation process of the peptide and the type of interaction between the cyclodextrin and the peptide. For the peptides reported in the literature, Trp and Phe are common preferential interaction sites with β -cyclodextrin (see Kitagawa (2015); Matilainen (2008); Ren (2016); and Qin XR, et al, Biochem Biophys Res Commun [ Biochemical and biophysical research communication ] 297: 1011-15 (2002)). The formation of inclusion complexes between cyclodextrins and aromatic residues may prevent intermolecular/intramolecular ii-ii interactions. The interaction of MEDI0382 with Trp and Phe was clearly demonstrated by near UV, Trp fluorescence and NMR analysis. Furthermore, after formulation in cyclodextrins, a profound change in secondary structure was observed, wherein the reduction of the alpha helix was compensated by the high beta-sheet content estimated for CD pro. This transformation suggests that the H-bond network that normally stabilizes the helical structure may be disrupted by preferential H-bonds between HP β CD and multiple amino acids when formulated in cyclodextrins. Since the assignment of peptide NMR has not been completely solved, the analysis of the interaction is limited to the assigned amino acids. Thus, a computational model was run to predict further interactions that occurred between HP β CD and MEDI 0382. Interestingly, simulations show that thermal movement of the lipid chain results in the formation of a complex with the cyclodextrin cavity, which is present throughout the simulation. Furthermore, analysis of the simulations revealed that a number of hydrogen bonding interactions occurred between HP β CD and several amino acid residues including aspartic acid (Asp), glutamic acid (Glu), and N-terminal histidine (His) (fig. 11). In addition to the hypothesis that secondary structure modification driven by hydrogen bonding is demonstrated, this observation also provides new insights about the fibrillation mechanism. The acidic residues Glu and Asp have pKa's as side chains of 3.9 and 4.0, respectively, whereas the N-terminal His has two basic groups, an alpha-amino group and an imidazolyl group. In glucagon having a structure similar to MEDI0382, the pKa of the 2 functional groups from His are reported to be 7.6 and 7.4, respectively (Hefford MA, et al, Biochemistry [ Biochemistry ]24 (4): 867-74 (1985)). Thus, at pH 7, Glu and Asp are both negatively charged, while His is positively charged, which can promote fibril formation through electrostatic interactions. However, inclusion complexes with charged residues may sterically hinder self-assembly when MEDI0382 is formulated in cyclodextrins. Finally, although the role of lipid chains in the aggregation process is not well understood, interactions with cyclodextrins as demonstrated by NMR, ITC and simulations may create further steric hindrance to self-assembly.

Example 6: effect of pH on Enteroinsulin peptides Co-formulated with DPZ in Cyclodextrin

To evaluate the effect of pH on the incretin peptide with DPZ in cyclodextrin, co-formulations were evaluated at pH 6.5 and 8 using (i) intrinsic trp fluorescence, (ii) Circular Dichroism (CD) before and after Tht, and (iii) TEM and Atomic Force Microscopy (AFM) photographs after Tht assay.

Formulations of MEDI0382 at pH 6.5 and pH 8 were compared using intrinsic Trp fluorescence in the presence and absence of cyclodextrin. In the absence of cyclodextrin, an increase in pH from 6.5 to 8 correlates with a trp fluorescence red-shift from 344nm to 348nm, respectively. In contrast, trp λ max was measured at 346nm regardless of pH when formulated in cyclodextrin. In addition, the fluorescence intensity increased 2-fold (FIG. 12, left). Interestingly, the far UV CD spectra also showed significant structural modification when formulated in cyclodextrins, with loss of helicity decreasing to 18% -19%, which can be compensated by an increase in β -sheet and random coil (fig. 12, right).

At pH 6.5, the Tht spectrum showed a very short lag time followed by a 30 hour growth phase and then a plateau was reached (fig. 13). AFM pictures taken at the end of the assay confirmed the presence of fibers (fig. 14), and the far UV CD spectrum after Tht assay indicated a loss of helicity (fig. 15). When the pH was increased to 8, Tht fluorescence remained at the level of the buffer control (fig. 13), indicating no fiber formation, as evidenced by the absence of ordered aggregates on AFM pictures (fig. 14).

Interestingly, cyclodextrin was added at pH 6.5 and although fibrillation of MEDI0382 occurred rapidly in buffer (lag time 3 hours), cyclodextrin completely inhibited aggregation during the assay (fig. 13). The absence of fibers on the AFM pictures (fig. 14) and the absence of CD spectra before and after Tht assay (fig. 15) rule out the possibility of generating false negatives due to the presence of cyclodextrins, and confirm the inhibitory effect of the macrocyclic molecules.

Aggregation kinetics measurements were also performed at pH 6.5 and pH 8 using the co-formulation. Tht assay at pH 6.5 showed an increase in fluorescence at 75 hours for the co-formulation, which was not seen for MEDI0382 in cyclodextrin alone (fig. 16). However, the lag phase of the coformulation was longer compared to MEDI0382 in buffer, which means that fibrillation was still delayed (fig. 16). In contrast to pH 6.5, the co-formulation proved to be stable at pH 8 (fig. 16).

Similar experiments were also performed using liraglutide co-formulations. As measured in the Tht assay, cyclodextrin appears to reduce liraglutide fibrillation at pH 6.5 (fig. 17). Similar to MEDI0382, cyclodextrin changed the secondary structure of liraglutide at both pH 6.5 and pH 8, and CD at pH 6.5 after Tht confirmed the presence of fiber in the buffer and cyclodextrin formulations (fig. 18).

These results indicate that cyclodextrins can increase the stability of lipidated intestinal insulinotropic peptides at least at pH 6.5 to 8.

Example 7: co-formulation of MEDI0382 and DPZ in cyclodextrins to maintain potency

Although HP β CD enhances the physical stability of the peptide, loss of the α -helix may have a significant impact on the potency of the peptide. There are indeed several studies that demonstrate that the secondary structure of GLP-1 and GLP-1 analogues plays a fundamental role in binding to and activation of the corresponding receptors (see e.g. Donnelly d., Br J Pharmacol. [ british journal of pharmacology ] 166: 27-41 (2012)). More specifically, the alpha-helical structure appears to be a key factor in the affinity and potency of driver peptides (Adelhorst K, et al, J Biol Chem. [ J. Biol. Chem. ] 269: 6275-8 (1994)). Thus, the biological activity of MEDI0382 on GLP1 and the glucagon receptor was evaluated in vitro to assess the effect of the presence of cyclodextrins and DPZ on its agonist properties. CHO cells overexpressing human recombinant GLP-1 or glucagon receptor were evaluated for potency in vitro and activity was reported as EC50 values after measuring cAMP accumulation. As shown in fig. 19A and 19B, no change in EC50 was observed for either receptor regardless of formulation. In addition, it is noteworthy that neither vehicle nor DPZ in vehicle showed activity on the receptor. The co-formulations are compatible with the once daily dosing frequency of MEDI0382 and DPZ.

Finally, the performance of MEDI0382 in co-formulations was evaluated in vivo to evaluate the effect of cyclodextrin and the presence of DPZ (fig. 19C and 19D). The pharmacokinetics of MEDI0382 was studied in cyclodextrin alone or co-formulated with DPZ after subcutaneous injection in rats and compared to the pharmacokinetics of MEDI0382 in buffer. Plasma concentrations versus time profile, and related PK parameters for MEDI0382 are reported in fig. 19C and table 3. DPZ is shown in figure 19D.

Table 3: average PK parameter of MEDI0382

Cmax-maximum plasma concentration; tmax-time to maximum plasma concentration; AUC_infThe area under the plasma concentration time curve to infinity; SD-standard deviation

(1) Tmax is reported as median

MEDI0382 in buffer showed slow absorption kinetics with a maximum concentration reached 4 hours after injection. When formulated alone in cyclodextrin, Tmax decreased significantly (1 and 4 hours for HPB and buffer formulations, respectively) and Cmax was about 1.5 times higher than the buffer formulation. Similarly, the total exposure increased 1.2 fold in the presence of cyclodextrin. The presence of DPZ in the co-formulation group did not cause any further change in PK compared to MEDI0382 in cyclodextrin. The elimination phase of MEDI0382 remained similar in all three formulations, indicating that these formulations did not affect the elimination of MEDI0382, which is driven primarily by binding to albumin.

In addition, the PK of DPZ was unchanged in the presence of MEDI 0382.

These data indicate that co-formulation of MEDI0382 and DPZ in cyclodextrin can maintain stability and bioefficacy of MEDI0382 at GLP1 and the glucagon receptor in vitro and in vivo. Thus, for MEDI0382 and DPZ, these co-formulations are compatible with once daily dosing frequency.

Claims (24)

1. A liquid pharmaceutical composition comprising (i) a lipidated intestinal insulinotropic peptide, (ii) a sodium glucose co-transporter 2 inhibitor (SGLT2i) and (iii) a cyclodextrin.

2. The composition of claim 1, wherein the insulinotropic peptide is mono-lipidated.

3. The composition of claim 1 or 2, wherein the insulinotropic peptide is a GLP-1/glucagon dual agonist peptide.

4. The composition of any one of claims 1-3, wherein the insulinotropic peptide is MEDI0382, liraglutide, or somaglutide.

5. The composition of any one of claims 1-4, wherein the SGLT2i is dapagliflozin.

6. The composition of any one of claims 1-5, wherein the cyclodextrin is beta cyclodextrin, optionally wherein the beta cyclodextrin is hydroxypropyl-beta-cyclodextrin.

7. The composition of any one of claims 1-5, wherein the cyclodextrin is sulfobutyl ether cyclodextrin.

8. The composition of any one of claims 1-7, wherein the lipidated incretin peptide is present at a concentration of about 0.5 mg/mL.

9. The composition of any one of claims 1-8, wherein the SGLT2i is present at a concentration of about 17 mg/ml.

10. The composition of any one of claims 1-9, wherein the cyclodextrin is present at a concentration of about 7% w/v.

11. The composition of any one of claims 1-10, wherein the SGLT2i and the cyclodextrin have a stoichiometry of about 1: 1.

12. The composition of any one of claims 1-11, wherein the composition has a pH of about 6.5 to about 8 or about 7 to about 8, optionally wherein the composition has a pH of about 7.

13. The composition of any one of claims 1-12, wherein the composition has a volume of 1mL or less.

14. The composition of any one of claims 1-13, wherein the composition is for parenteral administration, optionally wherein the parenteral administration is subcutaneous administration.

15. The composition of any one of claims 1-14, wherein the composition contains inclusion complexes comprising the lipidated insulinotropic peptide, the SGLT2i, and the cyclodextrin.

16. The composition of any one of claims 1-15, wherein the composition does not comprise fibrils of the lipidated insulinotropic peptide.

17. The composition of any one of claims 1-16, wherein the composition does not reduce the affinity of the lipidated insulinotropic peptide for the GLP-1 receptor and/or the glucagon receptor.

18. The composition of any one of claims 1-17, wherein administration of the composition to a rat results in a lipidated incretin peptide Cmax of about 390ng/mL, a lipidated incretin peptide Tmax of about 1 hour, a lipidated incretin peptide half-life of about 5 hours, and/or a lipidated incretin peptide AUC of about 3500-4000ng.hr/mL_0-inf。

19. An injection pen comprising the composition of any one of claims 1-18, optionally wherein the injection pen delivers about 600 μ Ι _, of the composition.

20. A method of treating type 2 diabetes in a subject in need thereof, the method comprising administering to the subject the composition of any one of claims 1-18, optionally wherein the subject is overweight or obese.

21. A method of treating non-alcoholic steatohepatitis (NASH) or non-alcoholic fatty liver disease (NAFLD) in a subject in need thereof, the method comprising administering to the subject the composition of any one of claims 1-18, optionally wherein the subject is overweight or obese.

22. A method of reducing liver fat in a subject in need thereof, the method comprising administering to the subject the composition of any one of claims 1-18, optionally wherein the subject is overweight or obese.

23. The method of any one of claims 20-22, wherein the administering delivers to the patient about 10mg of the SGLT2i and/or about 300 μ g of the lipidated incretin peptide.

24. The method of any one of claims 20-23, wherein the administration is diet and exercise assistance.

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